The invention refers to infective clones of the turnip mosaic virus (TuMV) and to plant infectious viral vectors based on said infectious clones.
Infectious viral clones comprise a DNA molecule and some transcription regulating mechanisms, capable of synthesising a transcript which can infect plants of plant cells (protoplasts). Infectious clones, in themselves, constitute a very valuable tool in basic research in Virology and, besides, are the necessary starting element for the preparation of plant viral vectors.
Plant viruses show different types of genoma: single stranded DNA (for example, geminivirus), double stranded DNA (for example, pararetrovirus), positive polarity RNA, i.e., messenger direction (for example potyvirus), negative polarity RNA (for instance rabdovirus), double polarity RNA (for instance tospovirus) or double stranded RNA (for instance reovirus). Recombinant DNA technology initially allowed the development of infectious clones of plant viruses with DNA genoma (geminivirus and pararetrovirus). Afterwards, thanks to the development of reverse transcriptases infectious clones have been obtained from numerous plant virus groups with positive polarity RNA genoma [Boyer, J. C. and Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses. Virology, 198, 415-426]. Infectious clones from the following potyviruses have been described:
Sharka virus (PPV) [Maiss E., Timple U., Brisske Rode A., Lesemann D. E. and Casper R. (1992). Infectious in vivo transcripts of a plum pox potyvirus full length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter. Journal of General Virology, 73, 709-712; Riechmann J. L., Lain S. and Garcia J. A. (1990). Infectious in vitro transcripts from a plum box potyvirus cDNA clone. Virology, 177, 710-126];
Tobacco vein mottling virus (TVMV) [Domier L. L., Franklin K. M., Hunt A. G., Thoads R. E. and Shaw J. G. (1989). Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proceedings of the National Academy of Sciences USA, 86, 3509-3513];
Tobacco engraving virus (TEV) [Doija V. V., McBride H. J. and Carrington J. C. (1992). Tagging of plant potyvirus replication and movement by insertion of xe2x96xa1-glucuronidase into the viral polyprotein. Proceedings of the National Academy of Sciences USA, 89, 10208-10212];
Pea seedborne mosaic virus (PSbMV) [Johansen I. E., Dougherty W. G., Keller K. E., Wang D. and Hampton R. O. (1996). Multiple viral determinants affect seed transmission of pea seedborne mosaic virus in Pisum sativum. Journal of General Virology, 77, 3149-3154];
Peanut stripe virus (PStV) [Flasinki S., Gunasinghe U. B., Gonzales R. A. and Cassidy B. G. (1996). The cDNA sequence and infectious transcripts of peanut stripe virus. Gene, 171, 299-300];
Zucchini yellow mosaic virus (ZYMV) [Gal-On A., Antignus Y., Rosner A. and Raccah B. (1991). Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus, Journal of General Virology, 72, 2639-2643; Galon A., Meiri E., Huet H., Hua W. J., Raccah B. and Gaba V. (1995). Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus, Journal of General Virology, 76, 3223-3227];
Papaya ringspot potyvirus (PRSV) [Chiang C. H. and Yeh S. D. (1995). Construction of infectious in vitro transcripts of papaya ringspot potyvirus. 6th International Plant Virus Epidemiology Symposium, Ma""ale Hachamisha, Jerusalem, Israel, page 52]; and
Potato A virus (PVA) [Puurand V., Valkonen J., Makinen K., Rabenstein F. and Saarma M. (1996). Infectious in vitro transcripts form cloned cDNA of the potato A potyvirus. Virus Research, 40, 135-140.
The genoma of most of the plant viruses comprises one or several positive polarity RNA molecules. The infectious clones of plant viruses with RNA genoma basically comprise a complementary DNA (cDNA) to the viral genomic RNA and a promoter sequence linked to said cDNA. This promoter sequence can either be that of a bacteriophage, which allows the obtaining of viral RNA by in vitro transcription by the corresponding RNA polymerase, or else that of A plant functional gene, which allows, after the introduction of the DNA into the plants, in vivo production of the corresponding effective viral RNA.
The obtaining of an full length infectious clone from an RNA template (the viral genoma of an RNA virus) implies, in the first place, the use of a reverse transcriptase in order to make a first cDNA chain, in the second place, the synthesis of double stranded cDNA by means of a DNA polymerase, and, in the third place, the linking of different cDNA restriction fragments, although this third stage is not always carried out because some full length infectious clones have been prepared by means of the polymerase (RT-PCR) reserve transcriptionxe2x80x94chain reaction. The obtaining of a full length infectious clone from an RNA template is a complicated procedure which shows many difficulties since (i) a reading error in the polymerases may take place during either of the first two stages, and (ii) errors may also occur in the linking of the restriction fragments either by the presence of exonucleases or else by the presence of restriction fragments, small and difficult to detect, for the enzyme being used, entailing both kinds of errors the obtaining of full length clones which are not infectious [Boyer, J. C. and Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses. Virology, 198, 415-426]. Another factor which also has an influence is the precision of the linking of the promoter to the cDNA. The infectivity is very much decreased, even with 5xe2x80x2 extensions of 1 or 2 nucleotides, however, the 3xe2x80x2 extensions do not have such a big influence in the biological activity [Boyer, J. C. and Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses. Virology, 198, 415-426]. On the other hand, the handling of full length viruses is very complex due to the unstableness problems which they show.
One of the main applications for plant infectious viral clones is their use in the construction of viral vectors. A plant infectious viral vector consists, in general, of a infectious viral clone modified in such a way that it contains a heterologous nucleic acid sequence which is expressed after inoculating plants or plant cells with said vector.
Plant viral vectors are used to research molecular biology processes in plants and are useful as tools in order to study plant viral infections processes. Besides, plant viral vectors have been developed in order to substitute and/or insert genes and to show epitopes. Among the commercial applications of known plant viral vectors are the expression of the interpheron alphaD gene, the expression of alpha-tricosanthine (inhibitor of viral replication in the human immunodeffiency virus [HIV]), the expression of the peptide inhibiting the enzyme which converts angiotensine I, the presentation of epitopes of the flu virus, of HIV-1, of malaria epitopes, and the expression of antigenic peptides of the glosopeda virus and human rhinovirus 14 (Scholthof, H. B., Scholthof, K. B. G. and Jackson, A. O. (1996). Plant virus gene vectors for transient expression of foreign proteins in plants. Annual Review of Phytopathology, 34, 299-323]. On the other hand, plant infectious vectors represent an alternative to the obtaining of transgenic plants and show numerous advantages, among which are, for instance, high expression in a short period of time, the expression of genes which can make more difficult the regeneration or the growth of transgenic plants and the possibility of a transient expression.
Plant viruses have a limited and specific range of hosts. Most of the plant species are not infected by most viruses, i.e., each virus is capable of infecting a unique combination of plant species [Dawson, W. O. and Hilf, M. E. (1992. Host range determinants of plant viruses. Annual Review of Plant Physiology and Plant Molecular Biology, 43, 527-555]. This situation implies that, with the present knowledge level of the factors determining the host range, it is impossible to obtain universal plant viral vectors. Therefore, there exists the need of looking for new clones and plant infectious viral vectors which can be used to infect useful plants as models for the study of the expression of heterologous and epitope genes of pathogens. The invention provides some turnip mosaic virus infectious clones, as well as some viral vectors based on said infectious vectors, adequate for infecting plants of Arabidopsis thaliana (L.) Heynh.
The turnip mosaic virus, hereinafter TuMV (Turnip mosaic virus) belongs to the potyvirus family, which are flexuous viruses whose virons are made of only one positive polarity RNA molecule of approximately 10,000 nucleotides, and of approximately 2,000 copies of only one type of protein, the coat protein (CP). The RNA genoma has a 5xe2x80x2 area and another 3xe2x80x2 one which are not translated (of approximately 150 and 200 bases respectively) constituting the central part of only one open reading stage which, when is translated, gives rise to only one polyprotein which is processed by means of at least 3 proteases coded by the virus in 8-10-gene products. [Riechmann J. L., Lain S. and Garcia J. A. (1992). Highlights and prospects of potyvirus molecular biology. Journal of General Virology, 73, 1-16]. The full sequence of a Canadian isolate of TuMV has been published [Nicolas, O. and Laliberte, J. F. (1992). The complete nucleotide sequence of turnip mosaic potyvirus RNA. Journal of General Virology, 73, 2785-2793] as well as that of a Japanese isolate [Oshima K., Tanaka M. and Sako N. (1996). The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain. Archives of Virology, 141, 1991-1997].